Design, synthesis, and biological evaluation of chromone-based p38 MAP kinase inhibitors

Article abstract of DOI:10.1021/jm200818j

3-(4-Fluorophenyl)-2-(4-pyridyl)chromone derivatives were synthesized and evaluated as p38 MAP kinase inhibitors. Introduction of an amino group in the 2-position of the pyridyl moiety gave p38α inhibitors with IC₅₀ in the low nanomolar range (

Full text of DOI:10.1021/jm200818j

BRIEF ARTICLE
pubs.acs.org/jmc
Design, Synthesis, and Biological Evaluation of Chromone-Based p38
MAP Kinase Inhibitors
Christine Dyrager,† Linda Nilsson M€ollers,† Linda Karlsson Kj€all,† John Patrick Alao,‡ Peter Dinꢀer,†
Fredrik K. Wallner,† Per Sunnerhagen,‡ and Morten Grøtli*,†
†Department of Chemistry, Medicinal Chemistry, University of Gothenburg, SE-41296 G€oteborg, Sweden
‡Department of Cell and Molecular Biology, University of Gothenburg, Box 462, SE-405 30 G€oteborg, Sweden
S Supporting Information
b
ABSTRACT: 3-(4-Fluorophenyl)-2-(4-pyridyl)chromone derivatives were synthesized and evaluated as p38 MAP kinase
inhibitors. Introduction of an amino group in the 2-position of the pyridyl moiety gave p38α inhibitors with IC50 in the low
nanomolar range (e.g., 8a, IC50 = 17 nm). The inhibitors (8a and 8e) showed excellent selectivity profiles when tested on a panel of
62 kinases, as well as efficient inhibition (8e) of p38 signaling in human breast cancer cells.
’ INTRODUCTION
The mitogen-activated protein kinases (MAPKs) are essential
regulators for signal transduction pathways and play crucial
roles in cellular processes such as transcription, apoptosis, and
differentiation.1 The p38 MAP kinase is highly expressed in
severe invasive breast cancers and is involved in the regulation of
cytokine biosynthesis (IL-1 and TNFα), which is associated with
chronic inflammatory diseases such as rheumatoid arthritis,
Crohn’s disease, and inflammatory bowel syndrome.2ꢀ4 Several
small molecule p38α MAPK inhibitors have been shown to block
the production of cytokines in vitro and in vivo, e.g., pyridylimi-
dazole derivatives exemplified by SB203580 (Figure 1).5ꢀ7
Furthermore, several natural occurring flavanoids, which contain
a chromone framework, were recently reported as p38α inhi-
bitors.8 We have for a long time been working on the synthesis
and functionalization of chromone derivatives9ꢀ14 because of
Figure 1. Pyridylimidazole derivative SB203580 acts as a p38α inhi-
bitor by binding to the ATP binding site of p38α.
their designation as privileged structures in drug discovery.15
Hence, in this study we have investigated the use of a 2,3-diarylated
chromone scaffold as a starting point for designing p38α inhi-
Compounds that contain the vicinal 4-fluorophenyl/pyridyl
motif, e.g., SB203580 (Figure 1), are known to interact with
the ATP binding site of the p38α MAP kinase.7,18ꢀ26 A key
interaction for these compounds is a hydrogen bond between the
pyridin-4-yl nitrogen and the backbone NH group of Met109 in
the hinge area.7,19 Furthermore, to obtain selectivity, the 4-fluor-
ophenyl moiety is placed in a hydrophobic pocket (I), which is
guarded by a gatekeeper residue (Thr106).7,17 It was found that
3-(4-fluorophenyl)-2-(4-pyridyl)chromone derivatives could mi-
mic the same binding mode as SB203580 and that introduction
of amino functions in the 2-position of the pyridyl moiety could
provide an extra hydrogen bonding interaction to the hinge
region (Figure 2).
bitors by using molecular modeling to explore the plausible
binding mode in the ATP-binding site of p38α. In this paper we
report the design, synthesis, and biological evaluation of 3-(4-
fluorophenyl)-2-(4-pyridyl)chromone derivatives as potential
p38α inhibitors. In addition, the activity of two chromone
inhibitors was tested against 62 different kinases to investigate
the selectivity across the human kinome. We also demonstrate
that these inhibitors can prevent anisomycin-induced p38 activa-
tion and downstream signaling in a human breast cancer cell line.
’ RESULTS AND DISCUSSION
Structure-Based Design. Docking of diarylated 4-fluorophenyl/
pyridyl chromone derivatives into the ATP binding site of p38α
(PDB code 1A9U), using the Schr€odinger package (Glide XP
mode), was performed to find a suitable substitution pattern on
the chromone ring system and to study potential interac-
tions with the active site to obtain inhibitory activity.7,16,17
Furthermore, the R-group on the amine could interact with
a secondary hydrophobic pocket (II). However, according to
Received: June 23, 2011
Published: September 12, 2011
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
BRIEF ARTICLE
gave high and reproducible yields of 6c (94%). Subsequent Pd-
mediated Suzuki coupling was used for the introduction of the
4-fluorophenyl moiety in the 3-position of 6a,b. However, standard
Suzuki coupling protocols gave poor and very low yields of
products.36ꢀ39 Compounds 7a and 7b were obtained in reason-
able yields (47% and 62%, respectively) when using an oxygen-
promoted ligand-free procedure with PEG-400 as solvent. This
procedure has previously been reported in the literature as a useful
protocol for the reaction with aryl chlorides.40 The final target
compounds 8aꢀg were obtained via a BuchwaldꢀHartwig amina-
tion with various amines in the presence of palladium(II) acetate,
2-(dicyclohexylphosphino)biphenyl or 1,3-bis[diphenylphosphino)-
propane], and sodium tert-butoxide in toluene.41ꢀ43
Biological Evaluation. The inhibitory potency of 7a,b and
8aꢀg were evaluated using a commercial radiometric p38α assay
performed by Millipore KinaseProfiler.44 The results, summar-
ized in Table 1, showed that all compounds acted as inhibitors of
the p38α MAP kinase. Compound 7a showed moderate activity
(IC50 = 813 nM) which decreased when replacing the hydrogen
in the 2-position on the pyridin-4-yl moiety with a chlorine
(7b, IC50 = 1380 nM). This negative effect on the inhibitory
activity can be explained as a result of the electron withdrawing
properties of the chlorine that leads to less efficient hydrogen
acceptor properties of the pyridine nitrogen. Introduction of
secondary amino functions in the 2-position of the pyridyl moiety,
with the possibility of an extra hydrogen bonding interaction to the
hinge area, gave good inhibitory activities for most of the com-
pounds (8aꢀc and 8eꢀg, IC50 = 17ꢀ45 nM). In contrast, intro-
duction of a diamine, such as in 8d, gave decreased activity (IC50
=
761 nM), which unexpectedly suggests that a polar group in a
hydrophobic pocket is unfavorable for the inhibitory activity.
Two of the inhibitors, 8a and 8e (at 0.8 μM), were chosen for a
selectivity screen against a panel of 62 kinases, which were
selected to represent the complete human kinome and kinases
closely related to the p38 MAP kinase.44 Only three kinases,
p38α, p38β, and JNK3, were strongly inhibited (0ꢀ25% remain-
ing kinase activity) (for IC50, see Supporting Information), whereas
most of the other kinases in the selected panel were not at all or
barely affected by the inhibitors (76ꢀ100% remaining kinase
activity) (Supporting Information, Table S2 and Figure S1). These
results suggest that 8a and 8e have a very good selectivity profile
toward the p38 kinase isoforms (α and β) among other human
kinases and it is reasonable to believe that the selectivity for
the compounds are even higher at lower concentrations (below
0.8 μM).
Compound 8e was also used to evaluate the efficacy in a cell-
based assay with human derived MCF-7 breast cancer cells.
Anisomycin-induced activation of p38 signaling, as shown for the
p38 phosphorylation targets activating transcription factor 2
(ATF2) and heat shock protein 27 (HSP27), was inhibited by
doses as low as 0.5 μM, and maximal inhibition was observed at
10 μM (Figure 3). Interestingly, 8e also inhibits phosphorylation
of p38 itself. Importantly, this occurs without affecting the total
levels of the kinase (data not shown), ruling out an effect on
protein stability of the inhibitor as the mechanism. Furthermore,
a supplemental experiment showed that phosphorylation of
MKK3 and MKK6, the upstream activators of p38, was un-
affected by 8e (Supporting Information, Figure S2). Thus, the
loss of p38 phosphorylation, induced by 8e, does not appear to
result from the inhibition of upstream signaling.
Figure 2. (A) Suggested interactions between 2-(2-amino-4-pyridyl)-
3-(4-fluorophenyl)chromones and the ATP-binding site in p38α.
(B) Compound 8a docked into the ATP-binding site of p38α.
modeling studies, the choice of substituents on the amine did not
seem to have any definite significance, as all the tested R-groups,
with different size and properties (alkylic, cyclic and aromatic),
were orientated in the same fashion into the hydrophobic area.
Furthermore, the chromone carbonyl oxygen can interact via
hydrogen bonding to the side chain of Lys53 in the active site. On
the basis of the modeling studies, we decided to synthesize a
small series of nine chromone derivatives that could be expected
to bind efficiently to the ATP binding site of p38α.
Chemistry. The target compounds were synthesized from 20-
hydroxyacetophenone 1 and the appropriate acid chloride 2a,b via
esterification to yield esters 3a,bfollowed by a BakerꢀVenkataraman
rearrangement to obtain diketones 4a,b (Scheme 1).27ꢀ29 There-
after, efficient acid-promoted cyclization afforded the flavone
derivatives 5a,b.29 Initial attempts to introduce a halogen sub-
stituent (bromide or iodide) into the 3-position of 5a,b were
performed using standard conditions, e.g., bromine in pyridine,
bromine in acetic acid, or N-halosuccinimides (NBS or NIS) in
DMF. However, none of the reactions produced the desired
products.29ꢀ32 Other attempts using iodine and CAN in acet-
onitrile or iodine and silver trifluoroacetate in DMF were also
performed but unfortunately without any product formation.33,34
Finally, the screening for viable reaction conditions gave the
3-iodo flavone derivatives 6a,b using in situ generated LDA and
iodine in THF.35 However, the reaction was not reproducible
when using the 2-chloropyridine derivate 5b. Instead, microwave
assisted bromination of 5b using excess NBS (5 equiv) in DMF
Alone, 8a and 8e did not significantly affect the proliferation of
MCF-7 or MDA-MB436 cells (Supporting Information Figure 3).
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Journal of Medicinal Chemistry
BRIEF ARTICLE
Scheme 1. Synthesis of 2-((2-Amino)-4-pyridyl)-3-(4-fluorophenyl)chromones (8aꢀg)a
a Reagents and conditions: (a) pyridine, 0 °C f room temp, 2 h; (b) KOH, pyridine, 100 °C, 15 min, microwave heating; (c) HCl, acetic acid, 60 °C,
30 min, microwave heating or H2SO4, EtOH, 100 °C, 15 min, microwave heating; (d) LDA, I2, THF, ꢀ78 °C, 15 min or NBS, DMF, 80 °C, 60 min,
microwave heating; (e) 4-fluorophenylboronic acid, K2CO3, Pd(OAc)2, PEG-400, 60 °C, 90 min, microwave heating; (f) amine, NaOtBu, Pd(OAc)2,
2-(dicyclohexylphosphino)biphenyl or 1,3-bis(diphenylphosphino)propane, toluene, 130 °C, 90 min, microwave heating.
Table 1. Biological Activity of 7a,b and 8aꢀg against the p38
MAP Kinase
Figure 3. Compound 8e inhibits p38 activation and signaling in human
breast cancer cells. MCF-7 cells were preincubated with the indicated
doses of 8e for 30 min and then exposed to 10 μg/mL anisomycin for
another 30 min. Total cell lysates were resolved by SDSꢀPAGE.
Membranes were probed with antibodies directed against phosphory-
lated ATF2, p38, and HSP27. Tubulin was used as a loading control.
p38 activity has previously been shown to suppress sensitivity to
genotoxic agents in p53 negative cells.45
’ CONCLUSIONS
We have developed an efficient synthetic route for the pre-
paration of 2-(2-aminopyridin-4-yl)-3-(4-fluorophenyl)chro-
mones. Several of the synthesized compounds demonstrate a
strong inhibitory activity toward the p38α MAP kinase (e.g., 8a,
IC50 = 17 nm). Among them, 8a and 8e were shown to be
selective inhibitors of the p38 isoforms (α and β), and it was also
revealed that 8e inhibits p38 signaling in human cancer cells.
Furthermore, molecular docking suggests that the synthesized
chromone-based compounds bind into the ATP binding site of
the p38α MAP kinase in a similar fashion as earlier known p38α
inhibitors containing the vicinal 4-fluorophenyl/pyridyl motif,
Furthermore, neither compound enhanced sensitivity to aniso-
mycin or doxorubicin in MCF-7 cells. However, 8a and 8e appeared
to suppress the sensitivity of MDA-MB436 cells, harboring a p53
mutation, to doxorubicin. This observation was unexpected, since
e.g., SB203580 (Figure 1). In conclusion, the syntheses of chro-
mone-based compounds represent a promising starting point for
the development of novel potent small molecule inhibitors of the
p38α kinase.
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Journal of Medicinal Chemistry
BRIEF ARTICLE
’ EXPERIMENTAL SECTION
the Chemical Biology Platform at the Faculty of Science, University
of Gothenburg, Sweden, and the Knut and Alice Wallenberg
Foundation for financial support.
General. All reagents and solvents were of analysis or synthesis
grade. 1H and 13C NMR spectra were recorded on a JEOL JNM-EX 400
spectrometer at 400 and 100 MHz, respectively, in CDCl3. Chemical
shifts are reported in ppm with the solvent residual peak as internal
standard (CHCl3 δ 7.26, CDCl3 δ 77.0) The reactions were monitored
’ ABBREVIATIONS USED
MAP, mitogen activated protein; IC50, the half maximal inhibi-
tory concentration; MAPK, mitogen activated protein kinase;
ATP, adenosine triphosphate; NBS, N-bromosuccinimide; NIS,
N-iodosuccinimide; DMF, N,N-dimethylformamide; CAN, cerium-
(IV) ammonium nitrate; LDA, lithium diisopropylamide; THF,
tetrahydrofuran; PEG-400, polyethylene glycol 400; SDSꢀPAGE,
sodium dodecyl sulfate polyacrylamide gel electrophoresis; ATF2,
activating transcription factor; HSP27, heat shock protein 27
by thin-layer chromatography (TLC), on silica plated (silica gel 60 F254
,
E. Merck) aluminum sheets, detecting spots by UV (254 and 365 nm).
Flash chromatography was performed manually on Merck silica gel 60
(0.040ꢀ0.063 mm) or using a Biotage SP4 Flash instrument with
prepacked columns. Solvents THF and toluene were refluxed over sodium/
benzophenone and distilled into 4 Å molecular sieves. Melting points were
measured in a B€uchi melting point B-540 apparatus and are uncorrected.
Microwave reactions were carried out in a Biotage Initiator instrument with
a fixed hold time using capped vials. Purities of the assayed compounds were
established by reversed-phase analytical HPLC and were found to be >95%.
The HPLC analysis was performed on a Waters 2690 separations module
system using an Atlantis T3, C18 (5 μm, 4.6 mm ꢁ 250 mm) column and a
Waters 996 photodiode array detector operating at a wavelength between
210 and 400 nm. High-resolution mass spectrometry data (nanospray
FT-ICR-MS) were obtained from BioAnser, Sahlgrenska Science Park,
Gothenburg, Sweden. Elemental analyses were performed at Kolbe
Mikroanalytisches Laboratorium, M€ulheim and der Ruhr, Germany.
General Procedure for the BuchwaldꢀHartwig Cross-
Coupling of Pyridyl Chlorides. Synthesis of 8bꢀg. The aryl
chloride (1.0 equiv), Pd(OAc)2 (0.05equiv),sodiumtert-butoxide (2.0 equiv),
and 1,3-bis(diphenylphosphino)propane (0.1 equiv) were added to a
dry microwave vial. The vial was evacuated and backfilled with nitro-
gen. Toluene (5 mL) was added to the vial, followed by the addition of
the appropriate amine (5.0 equiv). The mixture was heated at 130 °C
for 90 min in a microwave cavity. The mixture was diluted in
dichloromethane and filtered through a layer of Celite.
2-(2-(Butylamino)pyridine-4-yl)-3-(4-fluorophenyl)chromone
(8b). The title compound was synthesized according to general proce-
dure B. The crude product was purified by column chromatography
(heptane/ethyl acetate, gradient 20% f 40% ethyl acetate). Compound
7b (100 mg, 0.28 mmol) gave 8b (57 mg, 52%) as a yellow powder.
1H NMR (400 MHz, CDCl3) δ 0.94 (t, J = 7.3 Hz, 3H), 1.31ꢀ1.43
(m, 2H), 1.44ꢀ1.55 (m, 2H), 3.00ꢀ3.10 (m, 2H), 4.51ꢀ4.59 (m, 1H),
6.27 (s, 1H), 6.55 (dd, J = 1.4, 5.3 Hz, 1H), 7.00ꢀ7.11 (m, 2H), 7.19ꢀ
7.31 (m, 2H), 7.43ꢀ7.51 (m, 1H), 7.53ꢀ7.59 (m, m, 1H), 7.74 (ddd, J =
1.7, 7.2, 8.5 Hz, 1H), 8.04 (d, J = 5.3 Hz, 1H), 8.28 (dd, J = 1.6, 8.0 Hz,
1H); 13C NMR (100 MHz, CDCl3) δ 13.7, 20.1, 31.4, 41.9, 106.3, 112.1,
115.4 (d, 2JCF = 21.6 Hz, 2C), 118.0, 122.8, 123.4, 125.4, 126.4, 128.2
(d, 4JCF = 3.3 Hz, 1C), 132.7 (d, 3JCF = 8.1 Hz, 2C), 134.1, 141.9, 148.6,
155.9, 158.7, 160.0, 162.5 (d, 1JCF = 248.3, 1C), 177.2. HRMS (FT-ICR-MS):
[M + H]+ calcd for C24H22FN2O2: 389.1659. Found: 389.1658.
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’ ASSOCIATED CONTENT
S
Supporting Information. Synthesis of 2ꢀ8, compound
b
characterization, and biological procedures. This material is
available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: + 46 31 786 90 17. E-mail: grotli@chem.gu.se.
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